Peptides (v.43, #C)

Effects of 17β-estradiol on galanin(1-29)- and galanin(1-16)-like immunoreactivities by Jakob O. Strom; Tove Nilsson; Elvar Theodorsson (1-7).
► Galanin(1-16)-LI was affected by 17β-estradiol in brain and/or gut of rats. ► The pattern of galanin(1-16)-LI changes differed from that of galanin(1-29). ► Galanin(1-16)-LI could be a new member of the galanin neuropeptide family.There are reasons to believe that the galanin neuropeptide family could include more than the two hitherto known members (galanin(1-29) and galanin-like peptide), such as the existence of at least three galanin receptors and the fact that synthetic short-chain homologues have effects and binding sites that are distinct from those of galanin(1-29). The current study uses a radioimmunoassay based on a polyclonal rabbit antiserum raised against galanin(1-16) to study the concentrations of galanin(1-16) like immunoreactivity (LI) in the various parts of the brain and gut of ovariectomized female rats, and investigates the effects of different concentrations of estradiol on these concentrations in relation to galanin(1-29)-LI. Galanin(1-29) concentrations were increased by 17β-estradiol administration in almost all examined tissues whereas galanin(1-16)-LI was increased by 17β-estradiol treatment in most of the gut, but only in the pituitary of the brain. Furthermore, the relation between galanin(1-29)-LI and galanin(1-16)-LI varied substantially from tissue to tissue. The main hypothesis, that galanin(1-16)-LI would be affected by 17β-estradiol in brain and/or gut, was confirmed in addition to the secondary hypothesis, stating that the pattern of galanin(1-16)-LI changes would differ from that of galanin(1-29). The study indicates that galanin(1-16)-LI is estrogen-responsive but that its concentrations are regulated differently from that of galanin(1-29). This is strongly indicative of a biological relevance of this potentially new member of the galanin neuropeptide family.
Keywords: Galanin; Galanin(1-16); Neuropeptides; Rat; 17β-Estradiol; Dose–response relationship;

Elevated plasma visfatin concentrations in patients with community-acquired pneumonia by Wei Hu; Chang-Wen Liu; Jun Su; Jun Lu; Ying Zhu; Bing-Wei Liu (8-12).
► Plasma visfatin level increased in community-acquired pneumonia. ► Visfatin was associated with inflammation. ► Visfatin was associated with disease severity. ► Visfatin emerged as an independent predictor for 30-day mortality. ► Visfatin had high prognostic performance for 30-day mortality.Visfatin has been associated with some inflammatory disease. This study aimed to compare plasma visfatin levels in patients with community-acquired pneumonia and healthy controls and to furthermore investigate the relationship between their concentrations and 30-day mortality in patients. Plasma visfatin concentrations were measured in 176 patients and 95 healthy controls. The admission visfatin levels were significantly increased in all patients, survivals and non-survivals with community-acquired pneumonia compared with healthy control individuals, associated with pneumonia severity index score, Acute Physiology and Chronic Health Evaluation II score, white blood cell count, and plasma C-reactive protein level, and identified as an independent predictor for 30-day mortality. Its predictive value was similar to those of pneumonia severity index score and Acute Physiology and Chronic Health Evaluation II score. However, visfatin did not statistically significantly improve the predictive values of pneumonia severity index score and Acute Physiology and Chronic Health Evaluation II score. Thus, higher plasma visfatin level correlates with disease severity and markers of system inflammation and represent a novel biomarker for predicting 30-day mortality in patients with community-acquired pneumonia.
Keywords: Community-acquired pneumonia; Visfatin; Outcome; Mortality; Inflammation; Disease severity;

The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index by Miriam Goebel-Stengel; Tobias Hofmann; Ulf Elbelt; Pauline Teuffel; Anne Ahnis; Peter Kobelt; Nils W.G. Lambrecht; Burghard F. Klapp; Andreas Stengel (13-19).
► GOAT was recently detected in the circulation of rodents. ► GOAT protein is also present in human plasma. ► GOAT levels are lower in anorexia and higher in obesity compared to normal weight. ► Plasma GOAT is positively correlated with BMI and negatively with ghrelin. ► The ghrelin activating enzyme GOAT may play a role in anorexia and obesity.Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30–40, 40–50 and >50 were recruited (n  = 9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (−42%, p  < 0.01) whereas obese patients with BMI > 50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62–78%, p  < 0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r  = 0.71, p  < 0.001) and a negative correlation with ghrelin (r  = −0.60, p  < 0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.
Keywords: Anorexia; Body mass index; Circulation; Ghrelin; Hormone; Obesity; Plasma;

Exposure to chronic isolation modulates receptors mRNAs for oxytocin and vasopressin in the hypothalamus and heart by Hossein Pournajafi-Nazarloo; William Kenkel; Seyed Ramezan Mohsenpour; Lisa Sanzenbacher; Habibollah Saadat; Leila Partoo; Jason Yee; Fereidoun Azizi; C. Sue Carter (20-26).
► We examined the effect of repeated or chronic isolation on mRNAs for AVP and OT receptors in prairie voles. ► Chronic isolation increases plasma oxytocin in females and down-regulates OT receptor mRNAs in both sexes. ► Repeated isolation increased plasma AVP, which could be adaptive in response to stressor. ► The differential effects of isolation on OT and AVP systems help in understanding mechanisms through social interactions can be protective against emotional disorders.The goal of our study was to explore the effect of social isolation stress of varying durations on the plasma oxytocin (OT), messenger ribonucleic acid (mRNA) for oxytocin receptor (OTR), plasma arginine vasopressin (AVP) and mRNA for V1a receptor of AVP (V1aR) expression in the hypothalamus and heart of socially monogamous female and male prairie voles (Microtus ochrogaster). Continuous isolation for 4 weeks (chronic isolation) increased plasma OT level in females, but not in males. One hour of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma AVP level. Chronic isolation, but not repeated isolation, significantly decreased OTR mRNA in the hypothalamus and heart in both sexes. Chronic isolation significantly decreased cardiac V1aR mRNA, but no effect on hypothalamic V1aR mRNA expression. We did not find a gender difference within repeated social isolation groups. The results of the present study reveal that although chronic social isolation can down-regulate gene expression for the OTR in both sexes, the release of the OT peptide was increased after chronic isolation only in females, possibly somewhat protecting females from the negative consequences of isolation. In both sexes repeated, but not chronic, isolation increased plasma AVP, which could be permissive for mobilization and thus adaptive in response to a repeated stressor. The differential effects of isolation on OT and AVP systems may help in understanding mechanisms through social interactions can be protective against emotional and cardiovascular disorders.
Keywords: OT; AVP; Heart; Hypothalamus; Receptors; Stress; Social isolation;

The effects of adrenomedullin in traumatic brain injury by Hasan Demir; Ozge E. Onur; Arzu Denizbasi; Haldun Akoglu; Serkan E. Eroglu; Cigdem Ozpolat; Ebru Akoglu (27-31).
► Adrenomedullin exerts neuroprotective effects in animal models of acute brain injury. ► Adrenomedullin increased inflammatory markers in traumatic brain injury at dose of 12 μg/100 g in rats. ► In traumatic brain injury, adrenomedullin treatment significantly increased brain tissue MPO and MDA levels, decreased GSH levels.Traumatic brain injury (TBI) is a common cause of death and disability throughout the world. A multifunctional peptide adrenomedullin (AM) has protective effects in the central nervous system. We evaluated AM in an animal model as a therapeutic agent that reduces brain damage after traumatic brain injury. A total of 36 rats was divided into 3 groups as sham, head trauma plus intraperitoneal (ip) saline, and head trauma plus adrenomedullin ip. The diffuse brain injury model of Marmarou et al. was used. Blood samples were taken from all groups at the 1st, 6th and 24th hours for analysis of TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β) and IL-6 (interleukin-6) levels. At the end of the study (at the 24th hour) a neurological examination was performed and half of the rats were decapitated to obtain blood and tissue samples, the other half were perfused transcardiacally for studying the histopathology of the brain tissue. There were no statistically significant changes in plasma levels of IL-1β, IL-6 and TNF-α relative to the sham group. Also, changes in tissue levels of malonedialdehyde, myeloperoxidase and glutathione were not statistically significant. However, neurological scores and histopathological examinations revealed healing. AM individually exerts neuroprotective effects in animal models of acute brain injury. But the mechanisms of action remain to be assessed.
Keywords: Adrenomedullin; Traumatic brain injury; Myeloperoxidase; MDA; GSH; IL-6;

Brain natriuretic peptide predicts forced vital capacity of the lungs, oxygen pulse and peak oxygen consumption in physiological condition by Dejana Popovic; Miodrag C. Ostojic; Bojana Popovic; Milan Petrovic; Bosiljka Vujisic-Tesic; Aleksandar Kocijancic; Marko Banovic; Aleksandra Arandjelovic; Stanimir Stojiljkovic; Vidan Markovic; Svetozar S. Damjanovic (32-39).
► NT-pro-BNP has predictive value for heart rate. ► NT-pro-BNP has predictive value for forced vital capacity. ► NT-pro-BNP has predictive value for oxygen consumption. ► NT-pro-BNP has predictive value for oxygen pulse. ► NT-pro-BNP could be a common regulatory factor coordinating adaptation of heart and lungs to stress condition.Brain natriuretic peptide (NT-pro-BNP) is used as marker of cardiac and pulmonary diseases. However, the predictive value of circulating NT-pro-BNP for cardiac and pulmonary performance is unclear in physiological conditions. Standard echocardiography, tissue Doppler and forced spirometry at rest were used to assess cardiac parameters and forced vital capacity (FVC) in two groups of athletes (16 elite male wrestlers (W), 21 water polo player (WP)), as different stress adaptation models, and 20 sedentary subjects (C) matched for age. Cardiopulmonary test on treadmill (CPET), as acute stress model, was used to measure peak oxygen consumption (peak VO2), maximal heart rate (HRmax) and peak oxygen pulse (peak VO2/HR). NT-pro-BNP was measured by immunoassey sandwich technique 10 min before the test – at rest, at the beginning of the test, at maximal effort, at third minute of recovery. FVC was higher in athletes and the highest in W (WP 5.60 ± 0.29 l; W 6.57 ± 1.00 l; C 5.41 ± 0.29 l; p  < 0.01). Peak VO2 and peak VO2/HR were higher in athletes and the highest in WP. HRmax was not different among groups. In all groups, NT-pro-BNP decreased from rest to the beginning phase, increased in maximal effort and stayed unchanged in recovery. NT-pro-BNP was higher in C than W in all phases; WP had similar values as W and C. On multiple regression analysis, in all three groups together, ΔNT-pro-BNP from rest to the beginning phase independently predicted both peak VO2 and peak VO2/HR (r  = 0.38, 0.35; B  = 37.40, 0.19; p  = 0.007, 0.000, respectively). NT-pro-BNP at rest predicted HRmax (r  = −0.32, B  = −0.22, p  = 0.02). Maximal NT-pro-BNP predicted FVC (r  = −0.22, B  = −0.07, p  = 0.02). These results show noticeable predictive value of NT-pro-BNP for both cardiac and pulmonary performance in physiological conditions suggesting that NT-pro-BNP could be a common regulatory factor coordinating adaptation of heart and lungs to stress condition.
Keywords: Brain natriuretic peptide; Forced vital capacity; Oxygen pulse; Oxygen consumption; Cardiopulmonary test;

► PACAP stimulates the release of SL-α and SL-β from cultured goldfish pituitary cells. ► PACAP also enhances the expression of SL-β mRNA, but not SL-α mRNA. ► The action of PACAP is blocked by PACAP(6–38).Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10−10–10−7  M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10−9  M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC1R) antagonist, PACAP(6–38) (10−6  M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10−9 and 10−8  M) for 6 h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10−8  M) on SL-β mRNA expression was blocked by treatment with PACAP(6–38) (10−6  M), whereas PACAP(6–38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC1R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC1R signaling.
Keywords: Goldfish; PACAP; SL-α; SL-β; SL release; SL mRNA; Cultured pituitary cells; PAC1 receptor;

Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference by Amine Bahi; Virginie Tolle; Jean-Alain Fehrentz; Luc Brunel; Jean Martinez; Catherine-Laure Tomasetto; Sherif M. Karam (48-55).
► Ghrelin KO displayed lower ethanol-CPP. ► Ghrelin receptor antagonist JMV2959 reduced CPP-expression in C57BL/6 mice. ► Ghrelin KO displayed lower ethanol-induced locomotor stimulation. ► Ghrelin KO and JMV2959-injected mice showed reduced voluntary ethanol intake. ► JMV2959 reduced ethanol intake only in WT but not in ghrelin KO mice.Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist “JMV2959”. We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs.
Keywords: Addiction; Conditioned-place preference; Ethanol; Ghrelin; JMV2959; Locomotor activity; Two-bottle choice;

Expression of NPR-B in neurons of the dorsal root ganglia of the rat by Essam M. Abdelalim; Jean-Pierre Bellier; Ikuo Tooyama (56-61).
► We determine the expression of NPR-B in the rat DRG. ► NPR-B is highly expressed in small DRG neurons. ► NPR-B is co-localized with CGRP and IB4. ► NPR-B is present in some neuronal processes.C-type natriuretic peptide (CNP) is an abundant neuropeptide in the central nervous system, which exerts its physiological effects through natriuretic peptide receptor B (NPR-B). Recently, the CNP/NPR-B system has been recognized as an important regulator for the development of sensory axons. The dorsal root ganglion (DRG) contains neurons transmitting several kinds of spinal sensory stimuli to the central nervous system. In this study, we characterized NPR-B receptor expression in the rat DRG, using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Immunostaining revealed that NPR-B was expressed in neuronal cell bodies and processes of the DRG, with NPR-B immunoreactivity mainly prominent in small and medium-sized DRG neurons. Double-immunolabeling showed that NPR-B was expressed in calcitonin gene-related peptide- and isolectin B4-positive neurons. Furthermore, NPR-B expression was co-localized with calcitonin gene-related peptide in the dorsal horn of the spinal cord. Together, our data suggest that the natriuretic peptides may perform several biological actions on sensory neurons via their binding to NPR-B in the DRG.
Keywords: CG-B; CGRP; DRG; IB4; Immunohistochemistry; Sensory neurons;

Conformation propensities of des-acyl-ghrelin as probed by CD and NMR by Riccardo De Ricco; Daniela Valensin; Elena Gaggelli; Gianni Valensin (62-67).
Display Omitted► First structural analysis of des-acyl ghrelin in HFA–water solutions. ► CD, CSI and temperature coefficients support the occurrence of structured elements. ► NMR 3D structure shows a stable α-helix between Pro-7 and Gln-14. ► Des-acyl ghrelin and obestatin show similar α-helical structural features. ► Des-acyl ghrelin and obestatin may interact with the same competent receptor.Des-acyl-ghrelin is a 28 amino acid peptide secreted by both human and rat stomach. Together with ghrelin and obestatin, it is obtained by post-translational modification of a 117 aminoacid prepropeptide mainly expressed in distinct endocrine cell type in the stomach. Although its receptor has not been unambiguously identified so far, des-acyl-ghrelin is considered one of the strongest antagonists of ghrelin in activating the growth hormone secretagogue receptor (GHS-R). Here the secondary structure of des-acyl-ghrelin in different experimental conditions has been investigated and compared with that of obestatin, a bioactive peptide having similar biological functions. CD and NMR techniques have been combined for gaining the desired conformational features. The obtained structures support a steady alpha-helix structure spanning residues from 7 to 14, very similar to that observed for obestatin at the same experimental conditions, leading to suggest that a similar secondary structure can be associated with the similar biological role.
Keywords: Des-acyl-ghrelin; HFA; Alpha-helix; NMR; Structure; CD;

Therapeutic benefits of 9-amino acid peptide derived from prothymosin alpha against ischemic damages by Sebok Kumar Halder; Junya Sugimoto; Hayato Matsunaga; Hiroshi Ueda (68-75).
The purpose of study is to determine the minimum peptide sequence in prothymosin alpha that provides neuroprotection against ischemic stress. The major finding is that 9-amino acid peptide sequence P9 (a.a. 52–60) in ProTα rescues ischemia-induced cellular and functional damages in vitro and in vivo. Detailed about P9-induced neuroprotection may confer a therapeutic benefit for the treatment of ischemic damages.Prothymosin alpha (ProTα), a nuclear protein, plays multiple functions including cell survival. Most recently, we demonstrated that the active 30-amino acid peptide sequence/P30 (amino acids 49–78) in ProTα retains its substantial activity in neuroprotection in vitro and in vivo as well as in the inhibition of cerebral blood vessel damages by the ischemic stress in retina and brain. But, it has remained to identify the minimum peptide sequence in ProTα that retains neuroprotective activity. The present study using the experiments of alanine scanning suggested that any amino acid in 9-amino acid peptide sequence/P9 (amino acids 52–60) of P30 peptide is necessary for its survival activity of cultured rat cortical neurons against the ischemic stress. In the retinal ischemia-perfusion model, intravitreous injection of P9 24 h after ischemia significantly inhibited the cellular and functional damages at day 7. On the other hand, 2,3,5-triphenyltetrazolium chloride (TTC) staining and electroretinogram assessment showed that systemic delivery with P9 1 h after the cerebral ischemia (1 h tMCAO) significantly blocks the ischemia-induced brain damages. In addition, systemic P9 delivery markedly inhibited the cerebral ischemia (tMCAO)-induced disruption of blood vessels in brain. Taken together, the present study provides a therapeutic importance of 9-amino acid peptide sequence against ischemic damages.
Keywords: Blood vessel; Ischemia; Neuroprotective peptide; Prothymosin alpha;

In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice by Jie Wei; Xing Zhi; Xiao-lang Wang; Ping Zeng; Ting Zou; Bei Yang; Jing-lei Wang (76-82).
Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.
Keywords: Ghrelin; [d-Lys3]-GHRP-6; Naloxone; Antinociception; Tail withdrawal test; Hot-plate test; Growth hormone secretagogue receptor 1 alpha (GHS-R1a);

► The milk, cheese whey and plasma of dairy cows contain peptides. ► Cheese whey peptides are at a slightly higher level than milk peptides. ► Dairy cow milk peptides are generally at a higher concentration than in their plasma. ► Cheese whey peptides might be an important source for human diet. ► Adropin, nesfatin-1 ghrelins and salusins have now been found in the milk, cheese whey and plasma of dairy cows.Biological fluids (milk and serum/plasma) and cheese whey milk-derived fluid contain numerous molecules, especially amino acids and proteins. Therefore, the purpose of this study was to find out whether cheese whey (n:6), cow milk (n:6) and its blood (n  = 6) have adropin, nesfatin-1, apelin-12, ghrelins and salusin peptides. Adropin, nesfatin-1, apelin-12 concentrations were measured by ELISA, whereas ghrelin and salusin concentrations were measured by EIA methods. It was found that adropin, nesfatin-1, apelin-12, des-acylated ghrelin and salusins in cheese whey were higher than in the corresponding milk peptides and plasma of dairy cows, with the exception of salusin alpha and acylated ghrelin in milk being the same than that of the corresponding cheese whey concentration and plasma of dairy cows. A correlation was also found between milk peptides and cheese whey, as also with plasma of dairy cows. The data suggest that peptides in cow milk might be an important and nutritious food for (neonatal) calves and human diet due to their biological and physiological properties.
Keywords: Cheese whey; Adropin; Apelin-12; Ghrelins; Nesfatin-1; Salusins;

Age-dependent reduction of ghrelin- and motilin-induced contractile activity in the chicken gastrointestinal tract by Takio Kitazawa; Akiko Yoshida; Takuya Tamano; Hiroki Teraoka; Hiroyuki Kaiya (88-95).
► Age-dependent change in responses of ghrelin/motilin was examined in chicken. ► Contraction of ghrelin in proventriculus but not in crop decreased with age. ► Ghrelin receptor expression in proventriculus but not in crop decreased with age. ► Motilin receptor and contraction of motilin in proventriculus decreased with age. ► Age-related regulation of gastrointestinal motility by ghrelin/motilin is suggested.Ghrelin is an endogenous ligand for growth hormone secretagogue-receptor 1a (GHS-R1a) and stimulates gastrointestinal (GI) motility in the chicken. Since ghrelin stimulates GH release, which regulates growth, it might be interesting to compare ghrelin-induced responses in GI tract of different-aged chickens. Motilin is a ghrelin-related gut peptide that induces strong contraction in the small intestine. Aim of this study was to clarify age-dependent changes in ghrelin- and motilin-induced contractions of the chicken GI tract and expression of their receptor mRNAs. Chicken ghrelin caused contraction of the crop and proventriculus. Ghrelin-induced contraction in the proventriculus decreased gradually up to 100 days after hatching, but the responses to ghrelin in the crop were the same during the growth period. GHS-R1a mRNA expression in the crop tended to increase, but that in the proventriculus decreased depending on the age. Chicken motilin caused contraction of the chicken GI tract. Atropine decreased the responses to motilin in the proventriculus but not in the ileum. Motilin-induced contraction in the proventriculus but not that in the ileum decreased depending on post-hatching days. On the other hand, motilin receptor mRNA expression in every region of the GI tract decreased with age, but the decrease was more marked in the proventriculus than in the ileum. In conclusion, ghrelin- and motilin-induced GI contractions selectively decreased in the chicken proventriculus depending on post-hatching days, probably due to the age-related decrease in respective receptors expression. The results suggest an age-related contribution of ghrelin and motilin to the regulation of chicken GI motility.
Keywords: Ghrelin; Motilin; Chicken GI tract; Growth; Receptor expression; Contraction;

Assessment of antimicrobial peptide LL-37 as a post-exposure therapy to protect against respiratory tularemia in mice by Helen C. Flick-Smith; Marc A. Fox; Karleigh A. Hamblin; Mark I. Richards; Dominic C. Jenner; Thomas R. Laws; Amanda L. Phelps; Christopher Taylor; Sarah V. Harding; David O. Ulaeto; Helen S. Atkins (96-101).
Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.
Keywords: Antimicrobial peptide; LL-37; Francisella tularensis LVS; Mice;

Significant decrease in plasma midregional proadrenomedullin level in patients with end-stage renal disease after living kidney transplantation by Yosuke Suzuki; Hiroki Itoh; Fumihiko Katagiri; Fuminori Sato; Kanako Kawasaki; Yukie Sato; Yuhki Sato; Hiromitsu Mimata; Masaharu Takeyama (102-104).
Impaired renal function has been suggested to significantly impact plasma midregional proADM (MR-proADM) level. The aim of this study was to assess whether improvement of renal function after living kidney transplantation has an impact on plasma MR-proADM-like immunoreactive substance (IS) level. Eleven patients with end-stage renal disease (ESRD) who were scheduled to undergo the first living kidney allograft transplantation were enrolled. Plasma MR-proADM-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 days after kidney transplantation. Plasma MR-proADM-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma MR-proADM-IS level from before to 90 days after kidney transplantation (r s  = −0.70, p  < 0.0001). These results suggest that recovery of kidney function after kidney transplantation may lead to decrease in plasma MR-proADM level in patients with ESRD, and that plasma MR-proADM level may depend largely on renal function.
Keywords: Mid-regional pro-adrenomedullin; MR-proADM; Kidney transplantation; End stage renal disease; Adrenomedullin;

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions by Bálint Botz; András Imreh; Katalin Sándor; Krisztián Elekes; János Szolcsányi; Dóra Reglődi; John P. Quinn; James Stewart; Andreas Zimmer; Hitoshi Hashimoto; Zsuzsanna Helyes (105-112).
Display OmittedPituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK1 tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP−/−, Tac1−/−, and Tacr1−/−) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30–40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1−/− and Tacr1−/− mice, but was absent in PACAP−/− animals. Motor coordination of the PACAP−/− and Tac1−/− groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1−/− mice. Basal postoperative microcirculation on the plantar skin of PACAP−/− mice did not differ from the wildtypes, but was significantly lower in Tac1−/− and Tacr1−/− ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP−/− mice, but not in Tacr1−/− and Tac1−/− animals. Both PACAP and SP/NKA, but not NK1 receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications.
Keywords: Traumatic mononeuropathy; Sciatic nerve ligation; Mechanical hyperalgesia; Neurogenic vasodilation; Substance P; Tachykinin NK1 receptor;

► We firstly demonstrated the distribution sites of r/m HK-1 in mouse brain. ► The analgesia of r/m HK-1 is induced by the activation of NK1 receptor firstly. ► The analgesia of r/m HK-1 is also related to the expression increase of MOR and POMC.Hemokinin-1 is a peptide encoded by Pptc, which belongs to the family of mammalian tachykinins. Our previous results showed that rat/mouse hemokinin-1 (r/m HK-1) produced striking analgesia after intracerebroventricular (i.c.v.) injection in mice, and the analgesia could be blocked by the NK1 receptor antagonist and the opioid receptor antagonist, respectively. However, the precise distribution sites and the molecular mechanism involved in the analgesic effect after i.c.v. administration of r/m HK-1 are needed to be further investigated deeply. Using the fluorescence labeling method, our present results directly showed that r/m HK-1 peptides were mainly distributed at the ventricular walls and several juxta-ventricular structures for the first time. Our results showed that the mRNA expressions of NK1 receptor, PPT-A, PPT-C, KOR, PDYN, DOR and PENK were not changed markedly, as well as the protein expression of NK1 receptor was hardly changed. However, both the transcripts and proteins of MOR and POMC were up-regulated significantly, indicating that the analgesic effect induced by i.c.v. administration of r/m HK-1 is related to the activation of NK1 receptor first, then it is related to the release of endogenous proopiomelanocortin, as well as the increased expression level of μ opioid receptor. These results should facilitate further the analysis of the analgesia of r/m HK-1 in the central nerval system in acute pain and may open novel pharmacological interventions.
Keywords: Fluorescent labeled peptides; Rat/mouse hemokinin-1; Intracerebroventricular administration; Transcriptional expression; Protein expression;

Plasma intermedin levels in patients with acute myocardial infarction by Zhengbing Lv; Kai Wu; Xiaoping Chen; Xin Zhang; Biying Hong (121-125).
It has been shown that adrenomedullin (ADM) may function as a cardiovascular-regulatory peptide in humans. Intermedin (IMD) is a newly discovered peptide related to ADM and has a greater range of biological effects on the cardiovascular in animal experiments. The purpose of the study was to investigate the pathophysiological role of IMD in patients with acute myocardial infarction (AMI). The present study included twenty patients with acute ST-segment elevation myocardial infarction (STEMI), thirty-three with stable coronary heart disease (SCHD), and eighteen healthy controls. Plasma levels of IMD, malonaldehyde (MDA), and superoxide dismutase (SOD) and cardiac biomarkers were determined at one, two, four and seven days following AMI. Plasma IMD levels were significantly increased on day 1 in AMI patients when compared with SCHD subjects (P  = 0.014), and reached a peak of 181.88 ± 9.47 pg/ml at 96 h. Plasma IMD concentrations were correlated with MDA and SOD. Furthermore, patients with severe lesions in their coronary arteries tended to have higher plasma IMD levels (P  < 0.05) in AMI patients. A significant increase in plasma IMD following AMI may be associated with oxidative stress, and could be used as a marker to reflect the severity of the coronary stenosis.
Keywords: Intermedin; Acute myocardial infarction; Oxidative stress;

Purification, characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis by Chun-teng Guo; Stephen McClean; Chris Shaw; Ping-fan Rao; Ming-yu Ye; Anthony J. Bjourson (126-132).
► A novel chymotrypsin inhibitor, BBPTI-1, was found in Burmese Daboia russelii siamensis venom. ► BBPTI-1 showed strong chymotrypsin inhibition activity, but no trypsin inhibition activity. ► BBPTI-1 was cloned along with another cDNA with an eight nucleotide deletion.One novel Kunitz BPTI-like peptide designated as BBPTI-1, with chymotrypsin inhibitory activity was identified from the venom of Burmese Daboia russelii siamensis. It was purified by three steps of chromatography including gel filtration, cation exchange and reversed phase. A partial N-terminal sequence of BBPTI-1, HDRPKFCYLPADPGECLAHMRSF was obtained by automated Edman degradation and a Ki value of 4.77 nM determined. Cloning of BBPTI-1 including the open reading frame and 3′ untranslated region was achieved from cDNA libraries derived from lyophilized venom using a 3′ RACE strategy. In addition a cDNA sequence, designated as BBPTI-5, was also obtained. Alignment of cDNA sequences showed that BBPTI-5 exhibited an identical sequence to BBPTI-1 cDNA except for an eight nucleotide deletion in the open reading frame. Gene variations that represented deletions in the BBPTI-5 cDNA resulted in a novel protease inhibitor analog. Amino acid sequence alignment revealed that deduced peptides derived from cloning of their respective precursor cDNAs from libraries showed high similarity and homology with other Kunitz BPTI proteinase inhibitors. BBPTI-1 and BBPTI-5 consist of 60 and 66 amino acid residues respectively, including six conserved cysteine residues. As these peptides have been reported to have influence on the processes of coagulation, fibrinolysis and inflammation, their potential application in biomedical contexts warrants further investigation.
Keywords: Daboia russelii siamensis venom; Chymotrypsin inhibitor; Purification; Characterization; Cloning;

Prognostic value of leptin: 6-Month outcome in patients with intracerebral hemorrhage by Xin Zhang; Xiao-Min Lu; Li-Fa Huang; Xu Li (133-136).
Leptin has recently been discussed as a novel biomarker for the clinical outcome of critical illness. This study aims to investigate the prognostic value of leptin with regard to long-term clinical outcomes in patients with intracerebral hemorrhage. In 50 healthy controls and 92 patients with acute spontaneous basal ganglia hemorrhage presenting to the emergency department of a large primary care hospital, we measured plasma leptin levels using an enzyme-linked immunosorbent assay in a blinded fashion. Plasma leptin levels on admission were considerably higher in patients than healthy controls. A significant correlation emerged between plasma leptin level and National Institutes of Health Stroke Scale score. A multivariate analysis identified plasma leptin level as an independent predictor for 6-month clinical outcomes including 6-month mortality and unfavorable outcome (Modified Rankin Scale score > 2). Using receiver operating characteristic curves, we calculated areas under the curve for 6-month clinical outcomes. The predictive performance of leptin was similar to, but did not obviously improve that of National Institutes of Health Stroke Scale scores. Thus, leptin may help in the prediction of 6-month mortality and unfavorable outcome after intracerebral hemorrhage.
Keywords: Leptin; Intracerebral hemorrhage; 6-Month clinical outcome;

Caveolae-dependent internalization and homologous desensitization of VIP/PACAP receptor, VPAC2, in gastrointestinal smooth muscle by Sunila Mahavadi; Sayak Bhattacharya; Jennnifer Kim; Sally Fayed; Othman Al-Shboul; John R. Grider; Karnam S. Murthy (137-145).
The main membrane proteins of caveolae (caveolin-1, -2 and -3) oligomerize within lipid rich domains to form regular invaginations of smooth muscle plasma membrane and participate in receptor internalization and desensitization independent of clathrin-coated vesicle endocytosis. We have previously shown that Gs-coupled VIP/PACAP receptors, VPAC2, predominantly expressed in smooth muscle cells of the gut, are exclusively phosphorylated by GRK2 leading to receptor internalization and desensitization. Herein, we characterized the role of caveolin-1 in VPAC2 receptor internalization and desensitization in gastric smooth muscle using three approaches: (i) methyl β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae in dispersed muscle cells, (ii) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and (iii) caveolin-1 knockout mice (caveolin-1−/−). Pretreatment of gastric muscle cells with VIP stimulated tyrosine phosphorylation of caveolin-1, and induced VPAC2 receptor internalization (measured as decrease in 125I-VIP binding after pretreatment) and desensitization (measured as decrease in VIP-induced cAMP formation after pretreatment). Caveolin-1 phosphorylation, and VPAC2 receptor internalization and desensitization were blocked by disruption of caveolae with MβCD, suppression of caveolin-1 with caveolin-1 siRNA or inhibition of Src kinase activity by PP2. Pretreatment with VIP significantly inhibited adenylyl cyclase activity and muscle relaxation in response to subsequent addition of VIP in freshly dispersed muscle cells and in muscle strips isolated from wild type and caveolin-1−/− mice; however, the inhibition was significantly attenuated in caveolin-1−/− mice. These results suggest that caveolin-1 plays an important role in VPAC2 receptor internalization and desensitization.
Keywords: Caveolin-1; Endocytosis; G protein coupled receptor; GRK2;

The hypothalamic POMC mRNA expression is upregulated in prenatally undernourished male rat offspring under high-fat diet by Marie-Amélie Lukaszewski; Laura Butruille; Emmanuelle Moitrot; Valérie Montel; Anne Dickes-Coopman; Jean Lesage; Christine Laborie; Didier Vieau; Christophe Breton (146-154).
Epidemiological studies demonstrated that adverse environmental factors leading to intrauterine growth retardation (IUGR) and low birth weight may predispose individuals to increased risk of metabolic syndrome. In rats, we previously demonstrated that adult male IUGR offspring from prenatal 70% food-restricted dams throughout gestation (FR30) were predisposed to energy balance dysfunctions such as impaired glucose intolerance, hyperleptinemia, hyperphagia and adiposity. We investigated whether postweaning moderate high-fat (HF) diet would amplify the phenotype focusing on the hypothalamus gene expression profile. Prenatally undernourished rat offspring were HF-fed from weaning until adulthood while body weight and food intake were measured. Tissue weights, glucose tolerance and plasma endocrine parameters levels were determined in 4-month-old rats. Hypothalamic gene expression profiling of adult FR30 rat was performed using Illumina microarray analysis and the RatRef-12 Expression BeadChip that contains 21,792 rat genes. Under HF diet, contrary to C animals, FR30 rats displayed increased body weight. However, most of the endocrine disorders observed in chow diet-fed adult FR30 were alleviated. We also observed very few gene expression changes in hypothalamus of FR30 rat. Amongst factors involved in hypothalamic energy homeostasis programming system, only the POMC and transthyretin mRNA expression levels were preferentially increased under HF diet. Both elevated gene expression levels may be seen as adaptive mechanisms counteracting against deleterious effects of HF feeding in FR30 animals. This study shows that the POMC gene expression is a key target of long-term developmental programming in prenatally undernourished male rat offspring, specifically within an obesogenic environment.
Keywords: Fetal; Maternal undernutrition; Appetite regulation; Microarray; Arcuate nucleus;

Association of an oral formulation of angiotensin-(1–7) with atenolol improves lipid metabolism in hypertensive rats by Cynthia F.F. Santos; Sérgio H.S. Santos; Adaliene V.M. Ferreira; Leida M. Botion; Robson A.S. Santos; Maria Jose Campagnole-Santos (155-159).
The β-adrenergic blockers and antagonists of the renin-angiotensin system (RAS) are among the drugs that present better results in the control of cardio-metabolic diseases. The aim of the present study was to evaluate the effect of the association of the β-blocker, atenolol, and an oral formulation of Ang-(1–7) on lipid metabolism in spontaneously hypertensive rats (SHR). The main results showed that SHR treated with oral formulation of Ang-(1–7) in combination to atenolol have an improvement of lipid metabolism with a reduction of total plasma cholesterol, improvement of oral fat load tolerance and an increase in the lipolytic response stimulated by the β-adrenergic agonist, isoproterenol, without modification of resting glucose or insulin sensitivity in adipocytes. In conclusion, we showed that administration of an Ang-(1–7) oral formulation in association with a β-blocker induces beneficial effects on dyslipidemia treatment associated with hypertension.
Keywords: Angiotensin-(1–7); Mas receptor; Atenolol; Dyslipidemia; SHR; Renin-angiotensin system;

Ghrelin and glucagon-like peptide-2 increase immediately following massive small bowel resection by Mitsuru Muto; Tatsuru Kaji; Motoi Mukai; Kazuhiko Nakame; Takako Yoshioka; Akihide Tanimoto; Hiroshi Matsufuji (160-166).
Children with short bowel syndrome face life-threatening complications. Therefore, there is an urgent need for a new therapy to induce effective adaptation of the remnant intestine. Adaptation occurs only during feeding. We focused on preprandial acyl ghrelin and des-acyl ghrelin, and postprandial glucagon-like peptide-2 (GLP-2), which are known to have active orexigenic and trophic actions. This study aims to clarify the secretion trends of these hormones after massive small bowel resection and to obtain basic data for developing a new treatment. Sixty-three growing male rats were used: 3 were designated as controls receiving no operation and 60 were randomized into the 80% small bowel resection (80% SBR) group and the transection and re-anastomosis group. Changes in body weight, food intake, and remnant intestine morphology were also assessed for 15 days after the operation. Acyl ghrelin and des-acyl ghrelin levels increased immediately, equivalently in both operation groups (P  = 0.09 and 0.70). Interestingly, in 80% SBR animals, des-acyl ghrelin peaked on day 1 and acyl ghrelin peaked on day 4 (P  = 0.0007 and P  = 0.049 vs controls). GLP-2 secretion was obvious in 80% SBR animals (P  = 2.25 × 10−6), which increased immediately and peaked on day 4 (P  = 0.009 vs. controls). Body weight and food intake in 80% SBR animals recovered to preoperative levels on day 4. Morphological adaptations were evident after day 4. Our results may suggest a management strategy to reinforce these physiological hormone secretion patterns in developing a new therapy for short bowel syndrome.
Keywords: Acyl ghrelin; Des-acyl ghrelin; GLP-2; Adaptation; Massive small bowel resection; Short bowel syndrome;

The effects of oral delivery of exenatide or pramlintide acetate in dodecyl maltoside (DDM) on energy balance and glycemic control in insulin-resistant obese db/db mice are enhanced when given in combination with [D-Leu-4]-OB3. To examine the anti-hyperglycemic influence of [D-Leu-4]-OB3 in a non-obese insulin-deficient animal model, we compared the effects of metformin (200 mg/kg) and [D-Leu-4]-OB3 (40 mg/kg) on energy balance and glycemic control in streptozotocin (STZ)-induced diabetic male Swiss Webster (SW) mice. Diabetic mice were given insulin (Levemir®, sc) alone, or in combination with metformin or [D-Leu-4]-OB3 orally in DDM, for 14 days. Body weight and food and water intake were measured daily. Fasting blood glucose levels were determined every other day. Serum C-peptide was measured by ELISA. Diabetic mice receiving insulin alone for 14 days gained significantly more weight than DDM-treated control mice, or mice given insulin in combination with metformin or [D-Leu-4]-OB3. The weight gain seen in mice given insulin alone was accompanied by significant increases in both food and water intake. Mice treated with insulin in combination with metformin or [D-Leu-4]-OB3, consumed significantly less food and water. Blood glucose levels in STZ-treated mice receiving insulin alone were reduced to 65.3% of initial levels, while mice receiving insulin with metformin or [D-Leu-4]-OB3 were reduced to 44.5% and 38.9%, respectively. Our results indicate that [D-Leu-4]-OB3 is as effective as metformin in preventing the body weight gain associated with insulin therapy, and on a molar basis, that the efficacy of [D-Leu-4]-OB3 as an insulin sensitizer may equal or surpass that of metformin.
Keywords: Diabetes; Obesity; Metformin; Insulin sensitizer; Leptin-related peptide;

Deciphering intracellular localization and physiological role of nociceptin and nocistatin by Saeed Tariq; Syed M. Nurulain; Kornélia Tekes; Ernest Adeghate (174-183).
► Nociceptin is a heptadecapeptide located in both the central and peripheral nervous systems. ► It regulates many functions including pain, memory and reproductive behavior. ► The electron microscopy has not been widely used in the intracellular localization of nociceptin.Nociceptin and nocistatin are endogenous ligands of G protein coupled receptor family. Numerous techniques have been used to study the diverse parameters including, localization, distribution and ultrastructure of these peptides. The majority of the study parameters are based on their physiological roles in different organ systems. The present study presents an overview of the different methods used for the study of nociceptin, nocistatin and their receptors. Nociceptin has been implicated in many physiological functions including, nociception, locomotion, stressed-induced analgesia, learning and memory, neurotransmitter and hormone release, renal function, neuronal differentiation, sexual and reproductive behavior, uterine contraction, feeding, anxiety, gastrointestinal motility, cardiovascular function, micturition, cough, hypoxic–ischemic brain injury, diuresis and sodium balance, temperature regulation, vestibular function, and mucosal transport. It has been noted that the use of light and electron microscopy was less frequent, though it may be one of the most promising tools to study the intracellular localization of these neuropeptides. In addition, more studies on the level of circulating nociceptin and nocistatin are also necessary for investigating their clinical roles in health and disease. A variety of modern tools including physiological, light and electron microscopy (EM) are needed to decipher the extent of intracellular localization, tissue distribution and function of these peptides. The intracellular localization of nociceptin and nocistatin will require a high resolution transmission EM capable of identifying these peptides and other supporting molecules that co-localize with them. A tracing technique could also elucidate a possible migratory ability of nociceptin and nocistatin from one cellular compartment to the other.
Keywords: Nociceptin; Nocistatin; Orphanin; Opiod; Investigative tools; NOP; OP4;